Method for virus propagation

ABSTRACT

The present invention relates to a method for virus propagation. More closely the invention relates to a method for animal component free propagation and production of rotavirus (RV) using recombinant trypsin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from United Kingdom patent applicationGB1900250.0, filed on Jan. 8, 2019, and United Kingdom patentapplication GB1905829.6, filed on Apr. 26, 2019. The entire content ofthe priority applications are fully incorporated by reference herewith.

Field of the Invention

The present invention relates to a method for virus propagation. Moreclosely the invention relates to a method for animal component freepropagation and production of rotavirus (RV) using recombinant trypsinfor virus activation and propagation.

BACKGROUND OF THE INVENTION

Rotavirus (RV) are intestinal pathogens that infect mostly infants andyoung children under five years with acute diarrhea. About 600 000children die every year from rotavirus, mainly in developing countries.The first efforts to develop a rotavirus vaccine began in the early1980s and two of the vaccines used today are Rotarix (GSK), which is amonovalent vaccine and RotaTeq (Merck), which is a pentavalent vaccine.To propagate human RV, cultivation has been done through the use ofprimary and transformed monkey kidney cells and by proteolyticactivation of the virus with trypsin prior to infection.

Today the RV production is done in T-flasks, cell factories or rollerbottles using Vero cells as a cell substrate and porcine trypsine foractivation of the RV. However, it is important to note that different RVstrains differ in their capacity to infect and replicate in cellculture. One of the most important things is to preserve the structureof the RV and to maintain all three protein layers of the virus particleto keep the RV infectious.

The virus protein (VP) have different roles in the replication cycle andone role is the activation step. This is done by trypsin cleavage ofVP4, that leads to virus penetration.

All current procedures for RV production require animal derived trypsinfor proteolytic virus activation. It would be desirable to have ananimal component free procedure and a new process for RV production inthe future.

SUMMARY OF THE INVENTION

The present inventors have provided an animal component free process forRV production using recombinant trypsin.

In a first aspect the invention relates to a method for rotavirus (RV)production comprising the following steps: cell cultivation in abioreactor, infection of the cells with RV by activation withrecombinant trypsin, and RV propagation within said bioreactor in thepresence of recombinant trypsin for maintenance/propagation.

The cell cultivation is preferably performed in VaccinExpress medium.

The bioreactor may be a T-flask or spinner flask or a disposable bag ona rocking platform, preferably a WAVE bioreactor, or a stirred tank.

Preferably the cell cultivation is performed on microcarriers. Mostpreferred the microcarriers are sterilized microcarriers, preferablyCytodex gamma.

The trypsin concentration is about 10 μg/ml for RV activation and about5 μg/ml for maintenance/propagation without microcarriers and about 20μg/ml for RV activation and about 7 μg/ml for maintenance/propagationwhen microcarriers are used.

Preferably the cells are Vero cells.

In a second aspect, the invention relates to use of the RV producedaccording to the method of the invention for formulation of a vaccineagainst rotavirus caused disease. Another use of the RV producedaccording to the method of the invention is for viral vector for genetherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of total RV production with the use of differentculture conditions, as detected by ELISA.

FIGS. 2a and 2b show ELISA and FFA results, respectively, indicatingimprovements of RV detection and infectious RV titer when theconcentration of recombinant trypsin was increased during the activationand maintenance step to support RV propagation.

-   -   1. Activation 10 ug/mL, Maintenance 3 ug/mL    -   2. Activation 10 ug/mL, Maintenance 5 ug/mL    -   3. Activation 10 ug/mL, Maintenance 7 ug/mL    -   4. Activation 20 ug/mL, Maintenance 7 ug/mL

FIGS. 3a and 3b . Microcarriers cultivation shows same cell growth andmorphology using OptiPRO SFM or VaccineXpress (FIG. 3a ) TOI (72 h)using OptiPRO SFM, (FIG. 3b ) TOI (72 h) using VaccineXpress.

FIG. 4. FFA results show that VaccineXpress promotes high RV infectioustiter.

-   -   1. OptiPRO SFM/recombinant trypsin    -   2. OptiPRO SFM/porcine trypsin    -   3. VaccineXpress/recombinant trypsin    -   4. VaccineXpress/porcine trypsin

FIG. 5. FFA results shows a successful RV infection when using animalfree components and Cytodex 1 Gamma in spinner flask cultivation.

-   -   1. OptiPRO SFM/porcine trypsin    -   2. VaccineXpress/recombinant trypsin

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described more closely in association withsome experiments below which were divided into two parts. The first partwas to perform a screening in T-flask, where a comparison betweenOPTIPRO SFM/VaccineXpress medium and porcine trypsin/recombinant trypsinwas done. During this part, trypsin concentration and time required toactivate the virus, was evaluated and optimized to improve RVpropagation.

The second part was to continue using Cytodex 1 Gamma microcarriers inspinner flask cultivation to repeat the same comparison betweendifferent medium and trypsin sources and finally a scale up processusing a ReadyToProcess WAVE25 Bioreactor was done to confirm an animalcomponent free process in single use equipment for virus production.

Terminology

Term Comment RV Rotavirus VP Viral Protein TC Tissue Culture CPECytopathic effect FBS Fetal Bovine Serum MA104 Monkey African Greenkidney cell MVB Master Virus Bank WVB Working Virus Bank WCB WorkingCell Bank FFU Fluorescence Focus Unit FFA Fluorescence Focus Assay ELISAEnzyme-linked immunosorbent assay MOI Multiplicity of Infection TOI Timeof Infection TOH Time of Harvest SFM Serum Free Medium PBS PhosphateBuffered Saline EDTA Ethylenediaminetetraacetic acid FITC Fluoresceinisothiocyanate MEM Minimal essential MediumMaterials/EquipmentChemicals, Reagent and Media

Description Cat. Number/Vendor MA104 Cells 85102918, ATCC Vero CCL-81,ATCC Rota virus VR-2018, ATCC/Sigma Aldrich MEM/EBSS SH30024.02(ABC212816), HyClone FBS SV30160.03 (RYL35916), HyClone OPTIPRO ™SFM12309-19 Thermo Scientific, Lot 1722698 VaccineXpress RR16207.01 LotRRH178741, HyClone Trypsin 25 g/L T4549-20ML (SLMB5253U), Sigma AldrichrTrypsin 1320024671, Kerry Sheffield DPBS (6x1L sterile) D8537 6x1LSigma Aldrich, lot RNBG0124 DPBS with Ca Mg (6x1L D8662 6x1L SigmaAldrich, Lot sterile) RNBG1602 Trypsin (0.25%) SH30042.02 (J160004)HyClone HyQtase SV30030.01 Lot J170015 BSA A7906 Sigma Aldrich PBS (CaMg) SH30264.1S HyClone PBS for staining procedure BE17-516Q Lonza 4%Formaldehyde solution 1.00496.5000 Sigma Aldrich pH 6.9 Triton X-100T9284, Sigma Aldrich Tween 20 P7949, Sigma Aldrich L-glutamineSH30034.01 HyClone Pluronic F-68 24040-032 Thermo Fisher SigmacoteSL2-100 ml, Sigma Aldrich TrypLE Select 1X 12563-029 Thermo FisherTrypsin inhibitor T6522 Sigma Aldrich PBS-EDTA D8537 Sigma Aldrich AntiRV Antibody C66130M Meridian Goat anti mouse Ab FITC 97022, AbcamRidascreen ® Rotavirus enzyme C0901 (EMM Life science) immunoassayHoechst 33342 135-1304 (BioRad)/62249 (Thermo Fisher Scientific)Working Virus Bank (WVB)

Rotavirus A (VR-2018TM from ATCC) was used which is a Wa (TC adapted)strain wherein the original source was from a patient with positive RVin diarrhoea stool. A Working Virus Bank (WVB) was created by growingMA104 (ATCC) cells grown in Minimal essential medium (MEM GE Healthcare)supplemented with 5% fetal bovine serum (FBS, GE Healthcare) and 4 mMglutamine in a 2-layer Cell Factory (CF, Nunc) until the cells reached acell density of 1.5-2×105 cells/cm2. The MA104 cells were washed once inserum-free media and the last wash was done in PBS with Ca2+ Mg2+ toremove all traces of the serum before virus infection. The dilution ofrotavirus (VR2018, 1:10), porcine trypsin concentration (10 ug/ml,(Sigma Aldrich) and time for activation (1 h, 37° C.) for the cleavageof the VP4 protein. Activated rotavirus was added to the CF, enough tocover the cell layer and the cells were infected for 1 h at 37° C. withgently mixing. Upon infection, growth medium was added containingadditional trypsin (5 ug/ml) to support virus infection. Dailymicroscopic examination of the infection was done to observe theoccurrence of cytopathic effect. The CPE may have a varying appearanceand can be described as refractile rounding, sloughing, cell clumpingfollowed by lysis. Virus material was harvested by freeze-thawing threetimes to release virus and improve the virus yield, followed bycentrifugation (2000 rpm for 10 minutes) to remove cellular debris fromthe harvest. The WVB was aliquoted in 50 ml Falcon tubes and stored at−80° C.

The virus material was measured using Fluorescent Focus Assay (FFA) toestimate the infectivity titer. Unfortunately the amount of infectious(triple layer) and noninfectious (double layer) particles are unknownbut can be confirmed by TEM (electron microscopy).

EXPERIMENT 1

Comparison Between Cell Culture Medium and Trypsin for RV PropagationUsing Vero Cells in T-Flask Cultivation

Initial experiments were done in T-flasks and designed to compare twodifferent cell culture media, (VaccineXpress and OPTIPRO™ SFM) and twodifferent trypsin (porcine and recombinant) in T25 cultures. Foractivation, the virus protein (VP) for Rota have different roles in thereplication cycle and one role is the activation step. This is done bytrypsin cleavage of VP4 (RV attachment protein), allowing for entry ofthe virus into the host cell.

To activate the RV from WVB, concentration of 10 μg/ml trypsin for bothporcine (Sigma Aldrich) and recombinant trypsin (KERRY Bio-Science) wasused and added to the virus mix and incubated for 1 h in a 37° C.watherbath.

Vero cells were seeded and at the time of infection (TOI) was reached,the Vero cells were washed twice in PBS with Ca2+ Mg2+ before theactivated RV was added to the T-flasks and the cells were infected for 1hour during gently mixing in CO2 incubator. After incubation theinoculum was diluted with medium supplemented with trypsin (1 ug/ml) tosupport virus propagation. When an equivalent appearance of CPE occurredbetween the T-flasks, RV material was harvested. The result showedcomparable results in cell growth regardless the medium, it shows whenseeding 5×104 cells/cm2 cells in T flasks, the time of infection (TOI)was reached after 48 hour and the viable cell density (VCD) was1.5-2×105 cells/cm2.

The use of recombinant trypsin for activation and RV propagation wasinvestigated in order to develop an animal origin-free RV productionprocess. For RV detection, ELISA (RIDASCREEN®, rotavirus enzymeimmunoassay) was used which detected VP 6 antigen in all culture and inFIG. 1 small difference in total RV production was shown which indicatesthat possibility of a process optimization of recombinant trypsin can beachieved.

Furthermore an experiment was done to improve the RV propagation byincreasing recombinant trypsin concentration where differentconcentration for the activation step and maintenance was tested. Foractivation of the RV, two concentrations of the recombinant trypsin wastested, 10 μg/ml and 20 μg/ml for 1 h at 37° C. T-25 flasks were seededand by time of infection (TOI) was reached the activated RV was added tothe T-flasks in a lower volume under gently mixing in CO2 incubator for1 hour and then the inoculum was diluted with medium using differentconcentrations of the recombinant trypsin (3, 5 and 7 μg/ml) to supportvirus propagation. When CPE occurs, RV material was harvested and RV wasmeasured by ELISA for RV detection and FFA to determine the infectiousvirus titer. This experiment using VaccineXpress medium indicate that RVtiter be improved by increasing recombinant trypsin concentration bothfor activation and maintenance to support RV propagation. Smalldifferences in the morphology and CPE during the RV infection could benoticed. Based on analytical results in FIGS. 2a and 2b , where anincrease in both RV detection (ELISA) and RV infectious particles(fluorescent focus unit/ml) was seen by increasing the concentration ofrecombinant trypsin. Parameters for recombinant trypsin concentrationwas set to 20 μg/ml for RV activation and 7 μg/ml for maintenance tosupport RV propagation.

EXPERIMENT 2

Comparison Between Cell Culture Medium and Trypsin for RV Propagationusing Vero Cells Culture on Cytodex 1 Gamma

Cytodex 1 Gamma was prepared for spinner flask using by the gammairradiated Cytodex 1 (3 g/L) in 50 ml complete cell culture medium andallowed to equilibrate in the incubator for at least 2 hours before theVero cells were added. Vero cells were detached from their cultivationflask, according to standard procedure Immediately after detachment,trypsin inhibitor (stock solution 1 mg/ml) was added to the cellsuspension, ⅕ of the volume of HyQtase that was used. The Vero cellswere counted and the start cell density was 0.3×106cells/ml, 15×106cells used for a 50-ml spinner flask cultivation using VaccineXpress orOptiPRO SFM supplemented with 4 mM L-Glutamine and 0, 2% Pluronic F-68.

The spinner flasks were placed on the stirrer platform (Techne) in theincubator (37° C., 5% CO2) under continuous stirring at 40 rpm.Approximately 50% of the medium was exchanged after 48 hours to maintaingood condition. Thereafter, cell growth was followed until the time ofinfection (TOI) and in FIG. 3 a VCD of 1×106 (+/−0.2×106) cells/ml wasreached. Samples were taken daily to determine the cell growth,viability and morphology. Cell concentration and viability wasdetermined using cell counter NC200.

For activation of the RV WVB, porcine trypsin (10 μg/ml) or recombinanttrypsin (20 μg/ml) were used for 1 h at 37° C. Vero cells were washedtwice in PBS with Ca2+ Mg2+ by letting the microcarrier settle and mediawas removed as much as possible without loosing any microcarriers. Afterthe last PBS washing step was done and removed, activated RV was addedto the spinner flask, ⅓ of the total culture volume and Vero cells wereinfected for 1 h in the incubator (37° C., 5% CO2) under continuousstirring at 40 rpm.

After the virus had infected the Vero cells, the inoculum was dilutedusing medium supplemented with either porcine trypsin (5 μg/ml) orrecombinant trypsin (7 μg/ml) to support virus propagation. Dailymicroscopic examination of infection was done and when CPE occurred, thematerial was harvested (TOH). RV material was transferred into a 50 mlconical tube and was freeze-thawed at −80° C. three times to improve thevirus yield, followed by low-speed centrifugation (2000 rpm for 10minutes) to remove cellular debris from the harvest lysate. Theclarified RV was transferred into a new 50 ml conical tube and stored at−80° C. RV material was analyzed by ELISA for RV detection and FFA todetermine the infectious virus titer.

Interestingly, the use of different culture conditions for RV infectioustiter seems to have a impact of RV propagation, in FIG. 4 showsdifference between the cell culture medias with up to ×10 moreinfectious particles produced in the VaccineXpress. This suggests thatthe cell culture medium composition is important for Rotavirusinfectivity under serum-free conditions. This was confirmed by doing anadditional RV infection which shows in FIG. 5 a successful RV infectionwhen using animal free components and Cytodex 1 Gamma in spinner flaskcultivation.

EXPERIMENT 3

RV Production Using Vero Cells on Cytodex 1 Gamma in Bioreactor Culture

A ReadyToProcess WAVE 25 bioreactor, connected to one ReadyToProcessCBCU controller, was equipped with tray 20 and one 10 L cellbag. Beforeinoculation, the 10 L cellbag bioreactor was inflated with air and amixture of VaccineXpress medium and Cytodex 1 Gamma microcarriers wastransferred to the bioreactor and equalized at 37° C. and 5% CO2. Anoffset pH calibration was conducted on the culture bag before cellinoculation. Cells were inoculated at a concentration of 0.4×106cells/mL in 2 L working volume and culture was controlled at pH 7.1, 37°C. and 5% CO2. Rocking motion was set to 100%.

At culture initiation, rocking speed was set to 6 rpm at an 8° angle thefirst 2 hours and then increased to 10 rpm at a 6° angle. Two hoursafter seeding, sample was taken to ensure that the cells had started toattach to the microcarriers. Thereafter, sampling was conducted every 24h to determine cell density and morphology. Prior to sampling, rockingspeed was temporarily increased for 1 min to 20 rpm to ensure ahomogenous solution. After 48 or 72 h, 50% of the working volume wasexchanged for fresh culture medium. When reaching a density ofapproximately 1 (+/− 0.2)×106 cells/mL (TOI), the cells were infectedwith activated RV. For activation of the RV from the WVB, recombinanttrypsin (20 μg/ml) was used for 1 h at 37° C.

Vero cells were washed twice in PBS with Ca2+ Mg2+ by letting themicrocarrier settle and media was removed as much as possible withoutlosing any microcarriers. When last PBS washing step was done andremoved, activated RV was added to the WAVE, ⅓ of the total culturevolume and Vero cells were infected for 1 h under continuous rocking.After the RV had infected the Vero cells, the inoculum was diluted usingmedium supplemented with recombinant trypsin (7 μg/ml) to support viruspropagation.

Daily microscopic examination of infection was done and when CPE hadoccurred, the material was harvested (TOH). RV material was aliquot andtransferred into flasks and were freeze-thawed at −80° C. three times,followed by low-speed centrifugation (2000 rpm for 10 minutes) to removecellular debris from the harvest lysate. The clarified RV wastransferred into a new 50 ml conical tube and stored at −80° C. RVmaterial was analyzed by ELISA for RV detection and FFA to determine theinfectious virus titer.

The results in the table show that scaling up seems to have no effect onRV production.

Sample FFU/ml ELISA (OD 450) Rotavirus 5.1*10⁵ 1.2 Rotavirus ref sample2.6*10⁵ 1.2

Based on the data from T flask and spinner flask culture, the aim was toshow scalability solution for RV production using microcarriers and ananimal component free materials. It was found that the Vaccine Xpressmedium formulation promotes the stability of the RV to remain infectiousin Bioreactor culture.

Analysis

ELISA (RIDASCREEN®, rotavirus enzyme immunoassay) for RV detection is aquantitative method.

The outcome is presented as a dilution factor, which is compared to apositive and negative control. The microwell plate is coated with amonoclonal antibody against VP6. This VP6 group is a specific antigenthat is found in all rotaviruses that cause disease in humans. Presenceof rotavirus in the sample, a sandwich complex will be formed consistingof immobilized antibodies, RV antigens, and antibodies conjugated withthe biotin-streptavidin-peroxidase complex.

Fluorescence Focus Assay (FFA) is an infectivity method.

RV was serial diluted into a 96 well plate with MA104 cells. After RVinfection, MA104 cells were labelled with an anti-RV antibody and aFITC-conjugated secondary antibody. Plates were scanned with the IN CellAnalyzer 2200 and RV infectious cells (green fluorescent cells) werecounted using IN Cell Analyzer 1000 Workstation, and a titer offluorescent focus forming units (FFU) per ml was calculated.

Conclusion

The results of the invention show that there were significantdifferences for infectious RV between the tested cell culture media andthat recombinant trypsin can be used to replace porcine trypsin for RVproduction.

Pre-sterilized microcarrier cultures yielded similar cell-specificproductivity as cells grown in static mode in T-flasks. Furthermore, thedescribed culture conditions were successfully implemented for an animalcomponent-free RV propagation in a single-use ReadyToProcess WAVE 25bioreactor system. It is known that animal strains used in laboratorymodels reach titers of 10⁷ to 10⁸ per mL compared to human adapted RVstrains which average titers one to three logs less than most animalstrains. In the described experiments, an RV titer of 10⁵ per mL wasreached for a human adapted RV strain. FIG. 5 shows the surprisingresult that VaccineExpress gave about 7 times better titer thanconventional medium.

The present inventors have shown that infectious Rotavirus can beproduced using animal origin-free raw materials. This is important froma quality and regulatory perspective as animal derived components cancontain adventitious agents. It is well known that bovine serum cancarry adventitious virus and there is also a concern around prions. TheRotarix vaccine was found to contain porcine circovirus and porcinecircovirus DNA could also be detected in the Rotateq vaccine. It isbelieved that this contamination was introduced due to the use ofporcine trypsin. It is not possible to reduce or inactivate adventitiousagents in live virus-based vaccines. Thus, these vaccines need to beproduced with raw materials and processes that minimize allpossibilities to contamination. Another source of adventitious agentscould be open processing of cell culture such as manual handling of cellfactories or roller bottles. Furthermore, the pooling of numerous rollerbottles or cell factories also make it more difficult to detectpotential contamination in one of the vessels before pooling. The use ofmicrocarriers and recombinant trypsin in the method for RV of thepresent invention enables RV production in closed bioreactors and meansthat a single batch can be harvested in high yield without the need ofpooling numerous vessels.

The invention claimed is:
 1. A method for rotavirus (RV) productioncomprising the following steps: cultivating cells in a bioreactor,infecting the cells with RV by activation with recombinant trypsin, andpropagating the RV within said bioreactor in the presence of recombinanttrypsin for maintenance/propagation, wherein the trypsin concentrationis about 10 μg/ml for RV activation and about 5 μg/ml formaintenance/propagation.
 2. The method according to claim 1, wherein thecell cultivation is performed in VACCINEXPRESS medium.
 3. The methodaccording to claim 1, wherein the bioreactor is a T-flask or spinnerflask.
 4. The method according to claim 1, wherein the bioreactor is adisposable bag on a rocking platform, preferably a Wave bioreactor, or astirred tank.
 5. The method according to claim 1, wherein the cellcultivation is performed on microcarriers.
 6. The method according toclaim 1, wherein the microcarriers are sterilized microcarriers,preferably Cytodex gamma.
 7. A method for rotavirus (RV) productioncomprising the following steps: cultivating cells in a bioreactor,infecting the cells with RV by activation with recombinant trypsin, andpropagating the RV within said bioreactor in the presence of recombinanttrypsin for maintenance/propagation, wherein the cell cultivation isperformed on microcarriers, and wherein the trypsin concentration isabout 20 μg/ml for RV activation and about 7 μg/ml formaintenance/propagation.
 8. The method according to claim 1, wherein thecells are Vero cells.
 9. Use of the RV produced according to claim 1 forformulation of a vaccine against rotavirus caused disease.
 10. Use ofthe RV produced according to claim 1 as a viral vector for gene therapy.